Antisense oligodeoxynucleotides (ODNs) provide a means to sequence specifically inhibit synthesis of distinct proteins within a cell. For a review reference is made to Uhlmann, E. and Peyman, A., (1990) Antisense oligonucleotides: a new therapeutic principle, Chem. Rev., 90, 543. The prior art is aware that by targeting mRNA sequences which code for proteins associated with disease (for example, viral proteins), antisense ODNs can have a therapeutic effect. The exquisite specificity of DNA:RNA hybridization is expected in the art to provide drugs with fewer toxic side effects. Although the antisense oligonucleotide therapeutic principle is very appealing from a theoretical viewpoint, the state of the art is that because of their high cost and low molar potency, these agents are currently not used as effective antiviral drugs. Moreover, the highly charged ODNs do not enter the cytoplasm of cells easily, and therefore many approaches have been taken in the prior art to improve delivery of ODN drugs across membrane barriers.
Antigens oligonucleotides are another class of sequence specific drugs which can inhibit protein synthesis. For a review reference is made to Moffat, A. S. (1991) Triplex DNA Finally Comes of Age, Science 252, 1374. Antigene ODNs bind to duplex DNA as a third strand and can inhibit transcription of mRNA. In theory, antigene ODN drugs should be more potent than antisense ODN drugs since there is only one genetic target (DNA). Currently this technology is limited by the number of gene targets which triple strand binding ODNs can recognize, but the field is rapidly advancing. The potency of antigene ODNs can be further enhanced by modification with functional groups that react with the duplex DNA target strands. Alkylating groups or cleaving groups which are targeted by antigene ODNs have the potential to permanently inactivate specific genes, thereby providing a rational base for curing disease. Since these ODNs act in the nucleus of cells, they must also be delivered across membrane barriers.
Protein binding oligonucleotides are another potential class of therapeutic. These are oligonucleotides that bind to specific proteins. Recently, it has been reported that single stranded ODNs can be isolated which bind to protein targets in a sequence specific manner and inhibit protein function. This is described in the reference article Bock, et al., (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin, Nature, 355, 564. Other examples of protein binding ODNs are homopolymers of phosphorothioates (Agrawal, S. Goodchild, J., Civiera, M. P., Thornton, A. H. Sarin, P. S. and Zamecnik, P. C., (1988) Oligodeoxynucleoside phophoramidates and phosphorothioates as inhibitors of human immunodeficiency virus, Proc. Natl. Acad. Sci, U.S.A., 85, 7079) or phosphorodithioates (Marshall, W. S., Beaton, G., Stein, C. A., Matsukura, M., and Caruthers, M. H. (1992) Inhibition of human immunodeficiency virus activity by phosphorothioate oligodeoxycytidine, Proc. Natl. Acad. Sci. U.S.A., 89, 6265) which have been shown to bind to viral reverse transcriptase and inhibit HIV replication. A problem encountered in connection with oligonucleotides which target intracellular proteins is delivery across cellular membranes.
Ribozymes are another class of oligonucleotides which can sequence specifically catalyze the hydrolysis of target RNA strands. For a description see Cech, T. R. (1987) The chemistry of self-splicing RNA and RNA enzymes, Science 236, 1532. The prior art has already proposed using these catalytic, RNA based "scissors" as therapeutic agents. The problem again is the delivery of the ribozyme oligonucleotides across cellular membranes.
In light of the foregoing it appears desirable to improve delivery of ODN drugs from the extracellular media (i.e. serum) into the cytosol of cells. Polyanionic ODNs cross membranes poorly and can be degraded by nucleases before reaching their ultimate site of action (the cytoplasm or nucleus of cells). Thus, poor bioavailability is a major reason for the low potency of ODN drugs. Therefore it is desirable to take advantage of endocytosis; an uptake pathway which cells use to bring macromolecules across the plasma membrane and deliver them to lysosomes. Lysosomes are low pH, membrane bound vesicles which contain the hydrolytic enzymes necessary to digest the concentrated macromolecules.
Further, it is desirable to improve the potency of ODN drugs by targeting them to specific tissue types. There has been a significant research effort in the prior art on design of cleavable linking groups to attach drugs to targeting ligands. The "flagship" targeting ligands for tissue specific targeting of drugs are monoclonal antibodies. These compounds can be "engineered" to bind to specific cell-surface receptors (antigens) which are rapidly endocytosed. Therefore, it is desirable to improve the potency of ODN drugs by increasing transport of ODNs across cellular membranes and further to improve potency by targeting the nucleic acid specific ODN drugs to cell specific receptors through choice of appropriate ligands. Thus "matched sets" of nucleic acid specific ODNs and tissue specific targeting ligands are expected to provide drugs with higher therapeutic index than traditional pharmaceuticals.